Covalently circularized nanodiscs for studying membrane proteins and viral entry

Nasr, Mahmoud L.; Baptista, Diego; Strauß, Michael; Sun, Zhenyu; Grigoriu, Simina; Huser, Sonja; Plückthun, Andreas; Hagn, Franz; Walz, Thomas; Hogle, James M.; Wagner, Gerhard

doi:10.1038/nmeth.4079

Cited by 235 publications

(302 citation statements)

References 29 publications

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“…Our results are remarkable in that the SMA-EA-based macro-nanodiscs spontaneously align in an external magnetic field enabling the well-established solid-state NMR based structural and dynamics studies on membrane proteins, in addition to the commonly used multidimensional solution NMR experiments on SMA-EA nanodiscs [2] and MAS experiments [26] macro-nanodiscs. The reported experimental results provide valuable insights into the properties, structural assembly and mechanism of formation of nanodiscs.…”

mentioning

confidence: 83%

Bioinspired, Size‐Tunable Self‐Assembly of Polymer–Lipid Bilayer Nanodiscs

et al. 2017

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Polymer-based nanodiscs are valuable tools in biomedical research that can offer a detergent-free solubilization of membrane proteins maintaining their native lipid environment. Here, we introduce a novel ~1.6 kDa SMA-based polymer with styrene:maleic acid moieties that can form nanodiscs containing a planar lipid bilayer which are useful to reconstitute membrane proteins for structural and functional studies. The physicochemical properties and the mechanism of formation of polymer-based nanodiscs are characterized by light scattering, NMR, FT-IR, and TEM imaging experiments. A remarkable feature is that nanodiscs of different sizes, from nanometer to sub micrometer diameter, can be produced only by varying the lipid-to-polymer ratio. As demonstrated, the small size nanodiscs (up to ~30 nm diameter) can be used for solution NMR studies whereas the magnetic-alignment of macro-nanodiscs (diameter of >~40 nm) can be exploited for solid-state NMR studies on membrane proteins.

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confidence: 83%

Bioinspired, Size‐Tunable Self‐Assembly of Polymer–Lipid Bilayer Nanodiscs

et al. 2017

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“…cNW11 circularized nanodiscs were prepared as described previously 31 . Purified rTRPV6* was incorporated into cNW11 (2.0 mg/ml) circularized nanodiscs using the procedure described above for the MS2N2 nanodiscs but with a molar ratio of 1:10:267 (rTRPV6* monomer:cNW11:lipid).…”

Section: Methodsmentioning

confidence: 99%

Opening of the human epithelial calcium channel TRPV6

McGoldrick

Singh

Saotome

et al. 2017

Nature

194

322

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Ca2+-selective transient receptor potential vanilloid subfamily member 6 (TRPV6) channels play a critical role in calcium uptake in epithelial tissues1–4. Altered TRPV6 expression is associated with a variety of human diseases5, including cancers6. TRPV6 channels are constitutively active1,7,8 and their open probability depends on the lipidic composition of the membrane, increasing significantly in the presence of phosphatidylinositol 4,5-bisphosphate (PIP2)7,9. We previously solved crystal structures of detergent-solubilized rat TRPV6 in the closed state10,11. Corroborating previous electrophysiological studies3, these structures demonstrated that the Ca2+ selectivity of TRPV6 arises from a ring of aspartate side chains in the selectivity filter that tightly binds Ca2+. However, it has remained unknown how TRPV6 channels open and close their pores for ion permeation. Here we present cryo-EM structures of human TRPV6 in the open and closed states. The channel selectivity filter adopts similar conformations in both states, consistent with its explicit role in ion permeation. The iris-like channel opening is accompanied by an α-to-π helical transition in the pore-lining S6 helices at an alanine hinge just below the selectivity filter. As a result of this transition, the S6 helices bend and rotate, exposing different residues to the ion channel pore in the open and closed states. This novel gating mechanism, which defines the constitutive activity of TRPV6, is unique for tetrameric ion channels and provides new structural insights for understanding their diverse roles in physiology and disease.

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“…More recently, we engineered covalently circularized nanodiscs (cNDs) where a phospholipid bilayer is surrounded by amphipathic helical polypeptides, derived from Apolipoprotein A1, that can be circularized by sortase A. 3 With this approach, very homogeneous, covalently closed nanodiscs have been engineered with diameters of ~ 8, 11, 15, and 50 nm. These cNDs not only serve as excellent tools for the studies of small- or medium-size membrane proteins 17–18 but could also act as a surrogate membrane to study the interactions of small virus particles with their membrane-bound receptors.…”

mentioning

confidence: 99%

DNA-Corralled Nanodiscs for the Structural and Functional Characterization of Membrane Proteins and Viral Entry

Zhao¹,

Zhang

Hogle

et al. 2018

J. Am. Chem. Soc.

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Here we present a modular method for manufacturing large-sized nanodiscs using DNA-origami barrels as scaffolding corrals. Large-sized nanodiscs can be produced by first decorating the inside of DNA barrels with small lipid-bilayer nanodiscs, which open up when adding extra lipid to form large nanodiscs of diameters ~45 or ~70 nm as prescribed by the enclosing barrel dimension. Densely packed membrane protein arrays are then reconstituted within these large nanodiscs for potential structure determination. Furthermore, we demonstrate the potential of these nanodiscs as model membranes to study poliovirus entry.

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Covalently circularized nanodiscs for studying membrane proteins and viral entry

Cited by 235 publications

References 29 publications

Bioinspired, Size‐Tunable Self‐Assembly of Polymer–Lipid Bilayer Nanodiscs

Bioinspired, Size‐Tunable Self‐Assembly of Polymer–Lipid Bilayer Nanodiscs

Opening of the human epithelial calcium channel TRPV6

DNA-Corralled Nanodiscs for the Structural and Functional Characterization of Membrane Proteins and Viral Entry

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